共查询到16条相似文献,搜索用时 187 毫秒
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目的 建立高纯度、高活性的虎血清IgG纯化方法。方法 用饱和硫酸铵沉淀虎血清得到IgG粗品;结合Hitrap Protein A亲和层析预装柱及阴离子交换层析法对粗品IgG进一步分离纯化,采用PAGE电泳和Western-Blot免疫印迹法鉴定IgG纯度和免疫活性。结果 80 mL虎血清亲和纯化得到84 mg IgG,阴离子交换层析纯化得到30 mg虎的IgG纯品。结论 建立了简便快速、纯度高、活性好的虎血清IgG的分离纯化方法,为虎血清IgG二级抗体的制备提供了高纯度、活性好的一级抗体免疫原。 相似文献
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目的 制备高纯度貉血清IgG和兔抗貉IgG抗血清,作为建立多种动物抗体检测技术的储备。方法 采用Hitrap Protein A亲和层析及盐析再沉淀法纯化貉血清IgG,通过PAGE电泳和Western-Blot免疫印迹法对IgG作纯度及免疫活性检测;常规免疫法制备兔抗貉IgG血清。结果 貉血清IgG与Protein G虽有较强的结合力,但同时也结合血清中其他杂蛋白;用二步纯化法可从5 mL貉血清中纯化IgG约7 mg,电泳和免疫印迹测定显示,IgG纯度大于95%,常规免疫法制备抗血清免疫双扩散效价达1∶32。结论 建立了可行的貉血清IgG的纯化方法和高效价的兔抗貉血清IgG抗血清,为貉血清IgG二级抗体酶联物的制备储备了资源。 相似文献
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目的 纯化黑线毛足鼠和金仓鼠血清IgG,制备兔抗金仓鼠和黑线毛足鼠血清IgG的抗血清。方法 用Hitrap Protein G亲和层析纯化黑线毛足鼠和金仓鼠血清IgG,经SDS-PAGE电泳鉴定纯度,标准免疫方法免疫兔子制备抗血清。结果 黑线毛足鼠和金仓鼠血清IgG对protein G有很高的亲合性,用Hitrap Protein G亲和层析纯化,得到高纯度黑线毛足鼠和金仓鼠IgG,利用纯化的IgG作抗原制备了高效价的抗血清,免疫双扩散测定效价达1∶32和1∶16。结论 证实黑线毛足鼠和金仓鼠IgG和Protein G具有很高的亲和性,Protein G亲和层析是纯化黑线毛足鼠和金仓鼠IgG有效的方法之一,制备了黑线毛足鼠和金仓鼠IgG的抗血清。 相似文献
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目的 纯化长爪沙鼠血清IgG,制备兔抗长爪沙鼠IgG抗血清。方法 采用Hitrap Protein G亲和层析预装柱来纯化长爪沙鼠血清IgG;通过SDS-PAGE电泳和Western-Blotting免疫印迹法对长爪沙鼠血清IgG进行纯度鉴定,免疫兔子制备抗血清。结果 7 mL长爪沙鼠血清纯化得到11 mg IgG;电泳和免疫印迹测定,IgG纯度大于95%;用纯化的IgG作抗原制备了兔抗血清,免疫双扩散测定效价达1∶32。结论 建立了长爪沙鼠血清IgG的纯化方法,制备了长爪沙鼠IgG抗血清,证实长爪沙鼠血清IgG和Protein G具有较高的亲和性。 相似文献
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目的:比较两种抗体纯化方法在分离纯化树鼩IgG抗体的应用,制备抗IgG的多克隆抗体及检测。方法:采用两种商品化IgG抗体纯化试剂盒分离树鼩血清IgG抗体,采用SDS-PAGE和蛋白定量测定提纯IgG。以树鼩IgG作为抗原,与等量弗氏完全佐剂(第一次)、弗氏不完全佐剂(第二次)混合皮下注射免疫兔,对分离血清进行多克隆抗体纯化及Western Blot检测及定量分析。结果:两种方法均能有效分离纯化树鼩IgG,在经过Montage PROSEP-A试剂纯化后的IgG在纯度和含量方面均优于Protein A/G Matrix试剂。通过纯化后的树鼩IgG免疫兔制备的抗IgG抗体能有效识别树鼩IgG。结论:纯化的树鼩IgG具有良好免疫原性,由此制备的抗体具有高度特异性。研究结果为利用树鼩作为实验动物提供了必要的实验基础。 相似文献
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采用改进的饱和硫酸铵盐析和DEAE-52纤维素层析两步法提纯猪血清IgG,并分析了IgG得率低的可能原因。利用Bradford蛋白浓度测定法、SDS—PAGE测得盐析粗提蛋白得率为12.96mg/mL血清,纯度为61.4%;DEAE层析第一峰蛋白纯度达95.7%,得率为3.9mg/mL血清。结果表明两步法能获得免疫实验要求电泳纯IgG,也发现明显分开的两个层析峰含有相同分子量成分IgG,这是纯化得率低的主因。 相似文献
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目的纯化蝙蝠血清IgG,制备兔抗蝙蝠IgG酶标抗体。方法采用亲和层析纯化法纯化蝙蝠血清IgG,SDS-PAGE电泳鉴定蝙蝠IgG纯度。免疫大白兔制备兔抗蝙蝠IgG抗血清,免疫双扩散法测定抗血清效价,亲和层析纯化法纯化抗血清IgG。用改良过碘酸钠标记法制备兔抗蝙蝠IgG酶标抗体,直接ELISA和Western blot法对兔抗蝙蝠IgG酶标抗体进行工作浓度测定。结果纯化的蝙蝠血清IgG,其SDS-PAGE测定纯度大于95%;免疫大白兔所制备的抗血清免疫双扩散效价为1∶64;用改良过碘酸钠标记法制备兔抗蝙蝠IgG酶标抗体,其直接ELISA和Western blot工作浓度分别为1∶12800和大于1∶2000。结论制备了蝙蝠血清IgG的抗血清和酶标抗体,为蝙蝠的血清学检测体系提供了技术和资源储备。 相似文献
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Kang SH Shin GW Palaksha KJ Shin YS Kim YR Lee EY Suh EH Huh NE Oh MJ Jung TS 《Fish & shellfish immunology》2006,20(3):295-304
The efficacy of protein A-horse radish peroxidase (HRP), as compared to that of mouse polyclonal antibody raised against purified Ig, in detection of black rockfish (Sebastes schlegeli Higendorf) immunoglobulin (Ig) was examined. Protein A affinity chromatography successfully purified Ig from black rockfish serum; the purified-Ig could be visualised as two protein bands (MW 70 and 25kDa) following resolution with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. In SDS-PAGE immunoblot profiles of the purified-Ig, the mouse polyclonal antibody recognised both the light chain and heavy chains of rockfish Ig, whereas protein A-HRP immunostained only the heavy chain of rockfish Ig. These results suggest that protein A-HRP may be used to detect rockfish antibody-antigen complexes in immunoassays. In a 2-DE immunoblot assay for exploring antigenic profiles of Lactococcus garvieae KG9408, protein A-HRP successfully detected specific antibodies to antigenic proteins of L. garvieae in the rockfish Ig. In addition, enzyme linked immunosorbent assay (ELISA) showed a high correlation between the results obtained for positivity of L. garvieae when protein A-HRP and the mouse polyclonal antibody-was used to analyse samples from 25 diseased rockfish. These results collectively indicate that protein A-HRP has a high affinity for Ig, and may be useful for new investigations into the humoral immune responses of rockfish. 相似文献
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Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau 总被引:2,自引:0,他引:2
Specific and total serum immunoglobulins were extracted by immunoaffinity, mannan-binding protein and Protein A affinity chromatography from southern bluefin tuna (Thunnus maccoyii Castelnau) immunised with rabbit IgG, and from non-immunised southern bluefin tuna. SDS-PAGE in 10% reducing gels revealed two heavy chains with molecular weights of approximately 74.6 +/- 1.3 kDa and 71.2 +/- 0.9 kDa, and two light chains with molecular weights of approximately 29 +/- 1.2 kDa and 28 +/- 1.0 kDa. Under non-reducing, but denaturing, conditions in 4% and 5% SDS-PAGE gels, a high molecular weight and a low molecular weight fraction were demonstrated. By gel filtration using Sephacryl HR 300 a molecular weight of 845 kDa, consistent with a tetramer, was obtained for the high molecular weight fraction, and a molecular weight of 168 kDa, consistent with a monomer, was obtained for the low molecular weight fraction. The extinction coefficient at A280 for the purified immunoglobulin (Ig) was determined to be 1.24. Tuna a-rabbit IgG Ig was reactive with all non-reduced mammalian IgG antigens tested, suggesting that common conformational antigenic determinants were recognised. 相似文献
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Maria Cristiane Martins de Souza Igor Tadeu Lazzarotto Bresolin Sonia Maria Alves Bueno 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(5-6):557-566
The ω-aminohexyl diamine immobilized as ligand on CNBr- and bisoxirane-activated agarose gel was evaluated for the purification of human immunoglobulin G (IgG) from serum and plasma by negative affinity chromatography. The effects of matrix activation, buffer system, and feedstream on recovery and purity of IgG were studied. A one-step purification process using Hepes buffer at pH 6.8 allowed a similar recovery (69–76%) of the loaded IgG in the nonretained fractions for both matrices, but the purity was higher for epoxy-activated gel (electrophoretically homogeneous protein with a 6.5-fold purification). The IgG and human serum albumin (HSA) adsorption equilibrium studies showed that the adsorption isotherms of IgG and HSA obeyed the Langmuir–Freundlich and Langmuir models, respectively. The binding capacity of HSA was high (210.4 mg mL?1 of gel) and a positive cooperativity was observed for IgG binding. These results indicate that immobilizing ω-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma. 相似文献
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Igor Tadeu Lazzarotto Bresolin Mariana Borsoi-Ribeiro Juliana Rodrigues Caro Francine Petit dos Santos Marina Polesi de Castro Sonia Maria Alves Bueno 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2009,877(1-2):17-23
Tris(2-aminoethyl)amine (TREN) – a chelating agent used in IMAC – immobilized onto agarose gel was evaluated for the purification of IgG from human serum by negative chromatography. A one-step purification process allowed the recovery of 73.3% of the loaded IgG in the nonretained fractions with purity of 90–95% (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). The binding capacity was relatively high (66.63 mg of human serum protein/mL). These results suggest that this negative chromatography is a potential technique for purification of IgG from human serum. 相似文献
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Involvement of a non-hormone-binding 90-kilodalton protein in the nontransformed 8S form of the rabbit uterus progesterone receptor 总被引:2,自引:0,他引:2
Nontransformed 8S progesterone receptor (8S-PR) was purified by hormone-specific affinity chromatography from rabbit uterine low-salt cytosol containing 20 mM molybdate. In the eluate obtained with radioactive progestin, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) showed the presence of several bands, including three that corresponded to approximately 90-, approximately 120-, and approximately 85-kDa proteins. None of these three proteins was found in the eluate of the affinity column when the molybdate-containing cytosol was chromatographed in the presence of nonradioactive progesterone ("mock purification"). Subsequent purification of the affinity eluate by DEAE-Sephacel chromatography gave a single radioactive receptor peak at 0.15 M KCl (approximately 20% yield, 19% purity on the basis of one binding site per approximately 100 kDa) with a sedimentation coefficient of 8.5 S. Silver staining after SDS-PAGE revealed that this purified 8S-PR fraction contained mainly the 120-, 90-, and 85-kDa proteins. [3H]R5020-labeled 8S-PR purified by DEAE-Sephacel column chromatography was UV irradiated, and after SDS-PAGE the 120- and 85-kDa proteins were revealed, but the 90-kDa protein was not.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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目的:利用昆虫细胞表达系统生产重组的人增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),并进行纯化和抗体结合特性鉴定。方法:以HeLa细胞逆转录的cDNA为模板,扩增人PCNA基因,并插入杆状病毒载体AcMNPV。利用昆虫细胞得到PCNA基因的重组杆状病毒。病毒感染细胞表达蛋白,联合镍柱亲和层析和离子交换层析获得高纯度的重组人PCNA蛋白。ELISA法测定抗体结合特异性。结果:以HeLa细胞cDNA为模板得到的基因序列同GenBank的人PCNA基因序列一致。草地贪夜蛾细胞(Spodoptera frugiperda,Sf9)表达重组人PCNA(recombinant human PCNA,rPCNA)的最佳感染值(MOI)和感染时间分别为0.05h和144h。rPCNA的产量高达110mg/L细胞,纯度95%。间接ELISA法检测抗体结合特性,rPCNA的敏感性和特异性分别为93.3%和85.0%。结论:建立了rPCNA的表达和纯化方法,获得了高效表达、高度抗体结合特异性的PCNA蛋白,该蛋白质能进一步开发为PCNA相关疾病的体外诊断试剂盒,具较大的应用价值。 相似文献